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DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein
Authors:Satoshi Hara  Tatsuya Nojima  Kohji Seio  Masasuke Yoshida  Toru Hisabori
Institution:1. Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan;2. Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan;3. Frontier Research Center, Tokyo Institute of Technology, Nagatsuta 4259-S2-3, Midori-ku, Yokohama 226-8503, Japan;4. Department of Life Science, Tokyo Institute of Technology, Nagatsuta 4259-J2-16, Midori-ku, Yokohama 226-8501, Japan;5. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075, Japan
Abstract:

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.
Keywords:NHS  N-hydroxysuccinimide  AMS  4-acetamido-4&prime  -maleimidylstilbene-2  2&prime  -disulfonic acid  PEG-Mal  polyethylene glycol-maleimide  ssDNA  single strand DNA  Trxh1  cytosolic thioredoxin h1 from Arabidopsis thaliana  PrxQ  chloroplast peroxiredoxin Q from A  thaliana  EF-G  elongation factor G from Synechocystis sp  PCC 6803  MDH  malate dehydrogenase  NTR  NADPH-thioredoxin reductase from A  thaliana  Ni-NTA  nickel-nitrilotriacetic acid  DTNB  5  5&prime  -dithiobis-2-nitrobenzoic acid
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